RNA tumor viruses typically contain an RNA genome of about 10.000 nucleotides (Bishop, J. M., Ann. Rev. Biochem., 47:35-88 (1978)). The structure of the RNA is unique in several aspects (Wang, L. H., Ann. Rev. Microbiol., 32:61-92 (1978)). Apart from three well defined structural genes, a sequence of about 600 nucleotides, which is generally referred to as C or common region, is present in almost all RNA tumor viruses studied to date. (Bishop, J. M., Ann. Rev. Biochem., 47:35-88 (1978); Wang, L. H., Ann. Rev. Microbiol., 32:61-92 (1978)). This sequence, whose function is not known, is located at the 3'-end of the RNA immediately adjacent to a Poly A sequence (Wang, L. H., Ann. Rev. Microbiol., 32:61-92 (1978). When the RNA is copied into double-stranded DNA by the virion-associated reverse transcriptase, a portion (approximately 300 base pairs) of the C region is repeated in the DNA to form two large terminal repeats (LTR). (Hsu, T. W., Sabran, J. L., Mark, G. E., Guntaka, R. V. and Taylor, J. M., J. Virol., 28:810-818 (1978); Shank, P. R., Hughes, S. H., Kung, H. J., Majors, J. E., Quintrell, N., Guntaka, R. V., Bishop, J. M. and Varmus, H. E., Cell, 15:1383-1895 (1978)). Upon integration into a host cell genome the same order of the linear DNA, including the large terminal repeats, is maintained (Hughes, S. H., Shank, P. R., Spector, D. H., Kung, H. J., Bishop, J. M., Varmus, H. E., Vogt, P. K. and Breitman, M. L., Cell, 15:1397-1410 (1978); Sabran, J. L., Hsu, T. W., Yeater, C., Kaji, A., Mason, W. S. and Taylor, J. M., J. Virol, 29:170-178 (1979)). In other words, the large terminal repeats are flanked by the host cellular sequences and the arrangement of the integrated viral genome is colinear with unintegrated linear viral DNA.
In virus-infected cells, the host cellular RNA polymerase II transcribes viral RNA so that as much as 5-10% (5.times.10.sup.3 to 10.times.10.sup.3 copies per cell) of the total poly A-containing RNA in the cell is virus-specific. (Rymo, L., Parsons, J. T., Coffin, J. M. and Weissman, C., Proc. Natl. Acad. Sci. USA 71:2782-2786 (1974); Hayward, W. S., J. Virol., 24:47-63 (1977)). This observation suggests the presence of some unusual structural feature or features in the DNA of RNA tumor viruses which allow selective binding of RNA polymerase. One possibility is that a strong promoter is present in the large terminal repeat of viral DNA.
In several systems, including bacteriophages fd (Heyden, B., Nusslein, C. and Schaller, H., Nature New Biol. 96: 9-12 (1972)); T7 (Pribnow, D. J., Mol. Biol. 99: 419-443 (1975)) and .lambda. (Maniatis, T., Ptashne, M., Backman, K., Kleid, D., Flashman, S., Jeffrey, A. and Maurer, R., Cell 5: 109-113 (1975)) and bacteria (Gilbert, W., Cold Spring Harbor Symp. Quant. Biol. 41: 193-205 (1976); Brown, K. D., Bennet, G. N., Lee, F., Schweingruber, M. E. and Yanofsky, C., J. Mol. Biol. 121: 153-177 (1978)), the promoter sites have been isolated from the DNA are sequenced. This has been achieved by using Escherichia coli RNA polymerase which recognizes and tightly binds to a promoter sequence on the DNA and renders it resistant to subsequent digestion by deoxyribonuclease. In all the cases studied so far the polymerase protects from nuclease digestion a fragment about 35-40 base pairs long. Nucleotide sequence analysis has revealed a greater degree of sequence conservation among various promoters (Doi, R. H., Bact. Rev. 41: 568- 594 (1977)).
There are several subgroups of avian tumor viruses (Vogt, P. K., in Comprehensive Virology (Frankel, H., Conrad, R. R. and Wagner, R. F., eds.), Vol. 9, pp. 341-455, Plenum Press, New York (1977)). In these viruses at least portions of the C region appear to be well conserved among several exogenous viruses (Vogt, P. K., in Comprehensive Virology (Frankel, H., Conrad, R. R. and Wagner, R. F., eds.), Vol. 9, pp. 341-455, Plenum Press, New York (1977)). In the virus-infected cells, the host cell RNA polymerase II transcribes viral RNA as shown by the sensitivity of transcriptions to .alpha.-amanitin (Rymo, L., Parsons, J. T., Coffin, J. M., and Weissman, C., Proc. Natl. Acad. Sci. U.S.A. 71: 2782-2786 (1974); Jacquet, M., Groner, Y., Monroy, G. and Hurwitz, J., Proc. Natl. Acad. Sci. U.S.A. 71: 3045-3049 (1974)). Previous studies have shown that avian myeloblastosis virus specific RNA can be transcribed by E. coli RNA polymerase in a reconstructed chromatin system from virus infected chick cells at levels comparable to that of infected whole cells (Monroy, G., Jacquet, M., Groner, Y. and Hurwitz, J., Cold Spring Harbor Symp. Quant. Biol. 39: 1033-1041, (1974)). This suggests that E. coli RNA polymerase does recognize a putative promoter site and is able to transcribe viral RNA. Recently it has been shown that calf thymus RNA polymerase II recognizes and binds to polyoma DNA at the same sites as that of E. coli RNA polymerase (Lescure, B., Dauget, C. and Yaniv, M., J. Mol. Biol. 124: 87-96 (1978)). Therefore, studies with RNA polymerase from E. coli should enable one to identify the site, if it exists on the avian sarcoma virus (ASV) DNA. As set forth hereinafter, such a site in ASV DNA has been located in a region corresponding to the first 50-60 nucleotide heterpolymeric region of RNA immediately adjacent to the 3'-end poly (A) sequence. Furthermore, this RNA polymerase binding site has been shown to function as a promoter of gene expression when cloned in E. coli.